DNA: The Genetic Material

1
DNA The Genetic Material

• Dr. Henry O. Ogedegbe
• Department of EHMCS

2
The Hammerling Experiment Cells Store Hereditary
Information in the Nucleus

• Where is hereditary information stored in the
  cell?
• The Danish biologist Hammerling cut cells into
  pieces to see which were able to express
  hereditary information
• He chose the green alga Acetabularia which grows
  up to 5 cm as a model organism for his
  experiments
• The genus Acetabularia have distinct foot, stalk
  and cap regions and the nucleus is located in the
  foot
• He amputated the stalk of some cells and the feet
  of others
• He found that when he amputated the cap, a new
  cap regenerated from the remaining portions of
  the cell

3
The Hammerling Experiment Cells Store Hereditary
Information in the Nucleus

• When the foot was amputated however, no new foot
  regenerated from the cap or the stalk
• He therefore hypothesized that the hereditary
  information resided within the foot of
  Acetabularia
• His hypothesis was tested by selecting
  individuals from two species of the genius
  Acetabularia which had different caps
• A. mediterranea has a disc shaped cap
• A. crenulata has a branched flower-like cap
• He grafted a stalk from A. crenulata to a foot
  from A mediterranea

4
The Hammerling Experiment Cells Store Hereditary
Information in the Nucleus

• The cap regenerated looked somewhat like the cap
  of A. crenulata
• He then cut the regenerated stalk and all
  subsequent caps were disc shaped like like A.
  mediterranea
• The experiment supported Hammerlings hypothesis

5
Transplantation Experiments each Cell Contains s
Full Set of Genetic Instructions

• Robert Briggs and Thomas King tested the
  hypothesis that that the nucleus is the
  repository of hereditary information
• They removed the nucleus from frog eggs and found
  out that without the nucleus, the egg did not
  develop
• When the nucleus was replaced, with one from a
  frog embryo cell, the egg developed into an adult
  frog
• While this experiment produced abnormal frogs
  modifications of the experiment by other workers
  produced satisfactory results

6
The Griffith Experiment hereditary Information
Can Pass between organisms

• Discovery of Transformation
• Griffith performed experiments in which he
  injected mice with virulent strain of
  streptococcus pneumoniae
• The infected mice all died.
• When he infected similar mice with mutant strains
  of S. pneumoniae that lacked the virulence factor
  the mice showed no ill effect
• When he infected mice with dead mutant strain of
  S. pneumoniae the mice remained healthy
• Similarly, infection of the mice with the R form
  of the bacteria produced no ill effect in the
  mice

7
The Griffith Experiment hereditary Information
Can Pass between organisms

• When he infected similar mice with a mixture of
  dead virulent bacteria and the R form some of the
  mice died
• The virulence factor had been transferred to the
  R form which transformed the coatless form to the
  virulent form

8
The Avery Experiment The Transforming Principle
Is DNA

• Avery and co-workers characterized the
  transforming principle
• They prepared a mixture of dead and coatless S.
  pneumoniae similar to what Griffith had done
• Then they removed all the proteins from the
  mixture
• Despite the removal of the proteins, the
  transforming activity of the mixture was not
  reduced
• The properties of the transforming principle
  resembled those of DNA in many respects

9
The Avery Experiment The Transforming Principle
Is DNA

• Analysis of the purified principle produced
  elements which agreed closely with DNA
• In an ultracentrifuge the transforming principle
  migrated like DNA
• Removal of lipids and proteins from the principle
  did not diminish its activity
• Protein digesting enzymes did not affect the
  principle
• The DNA digesting enzyme DNase destroyed all the
  transforming principle

10
The Hershey-Chase Experiment Some Viruses Direct
Their Heredity with DNA

• Hershey-Chase experiment involved bacteriophages,
  viruses that attack bacteria
• They employed the bacteriophage T2 which is a DNA
  virus
• They labeled the viral DNA with radioactive
  isotope of phosphorus 32P and the protein coat
  with radioactive sulfur 35S
• After the labeled viruses were allowed to infect
  the bacteria the bacterial cells were agitated
  violently
• This was designed to remove the protein coats of
  the infecting viruses from the surface of the
  bacteria

11
The Hershey-Chase Experiment Some Viruses Direct
Their Heredity with DNA

• The 32P label had transferred to the interior of
  the bacteria and viruses released subsequently
  contained the 32P label
• The hereditary information injected into the
  bacteria that specified new generation of viruses
  was DNA
• Thus the DNA is clearly the repository of
  hereditary information

12
The Frankel-Conrat Experiment Other Viruses
Direct Their Heredity with RNA

• Fraenkel-Conrat experimented with RNA viruses to
  determine how they reproduce
• They employed the tobacco mosaic virus and the
  Holmes ribgrass virus.
• They separated the RNA from the proteins and
  discovered that the RNA molecules were still
  infective whereas the protein molecules were not

13
The Chemical Nature of Nucleic Acid

• The DNA was discovered in 1969 by Friedrich
  Miescher four years after the publication of
  Mendels work
• He extracted a white substance from human cells
  and fish sperm
• The proportion of nitrogen and phosphorus in the
  substance was different from any previous
  substances
• This convinced him that he was dealing with a new
  substance
• Due to its slight acidity, it came to be known as
  nucleic acid

14
The Chemical Nature of Nucleic Acid

• The primary structure was elucidated in the 1920s
  by the biochemist P.A. Levene
• DNA contains three components which include the
  phosphate group, five carbon sugars, and
  nitrogenous bases
• The nitrogenous bases are purines adenine
  guanine and pyramidines thymine, and cytosine
• RNA contains uracil instead of thymine
• A nucleotide consist of a sugar attached to a
  phosphate group and a base

15
The Chemical Nature of Nucleic Acid

• The four carbon atoms and the oxygen atom form a
  five membered ring
• The carbon atoms are numbered 1 to 5 proceeding
  clockwise from the oxygen atom
• The prime symbol indicates that the carbon refers
  to a carbon in a sugar rather than a base
• The subunits are linked together by
  phosphodiester bonds
• The resultant two-unit polymer still has a free
  5 phosphate group at one end and a free 3
  hydroxyl group at the other end

16
The Chemical Nature of Nucleic Acid

• Chargaffs Analysis showed that the nucleotide
  composition of DNA molecules varies in complex
  ways
• This led to Chargaffs rules
• The proportion of A always equals that of T and
  the proportion of G always equals that of C
• There is always an equal proportion of purines (A
  and G) and pyramidines (C and T)

17
The three-Dimensional Structure of DNA

• The work of Rosalind Franklin involved X-ray
  crystallographic analysis of DNA
• This involved bombarding the DNA molecules with
  beams of X-rays
• Rosalind used DNA in the form of fibers in the
  laboratory of Maurice Wilkins
• The work of Rosalind led to the discovery of the
  double helix by Crick and Watson
• The double helix is stabilized by antiparallel
  strands one chain running 3 to 5 the other 5
  to 3

18
The Meselson-Stahl Experiment DNA Replication Is
semiconservative

• The basis for copying the genetic information is
  complementarity
• If the DNA molecule is unzipped one would need
  only to assemble the appropriate complementary
  nucleotides
• This would produce two daughter duplexes with
  the same sequence
• This form of DNA replication is called
  semiconservative because the sequence of the
  original duplex is conserved
• Each strand of the duplex becomes part of another
  duplex

19
The Replication Complex

• The DNA polymerase III plays a very essential
  part in gene DNA replication
• The polymerase III is a complex of 10 different
  kinds of polypeptide chains
• The enzyme is a dimer with two similar
  multisubunit complexes
• Polymerase III threads the DNA through the
  complex at the rate of 1000 nucleotides per
  second

20
The Replication Complex

• The two strand of DNA are assembled differently
• The polymerase III can add nucleotide only to the
  3 end of a DNA strand
• That means that replication occurs in the 5 to
  3 direction on a growing DNA strand
• The leading strand is built up by adding
  nucleotides continuously to it growing 3 end
• The lagging strand which elongates away from the
  replication fork is synthesized discontinuously
  as short segments

21
The Replication Complex

• These discontinuous segments are called the
  Okasaki fragments
• They are about 100 to 200 nucleotides long in
  eukaryotes and about 1000 to 2000 nucleotides
  long in prokaryotes
• The Okasaki fragment is synthesized by DNA
  polymerase III in the 5 to 3 direction
• The overall replication of the DNA is said to be
  semidiscontinuous

22
The Replication Process

• The replication of the DNA molecule takes place
  in five interlocking steps
• Opening of the DNA double helix
• Initiation replication
• Unwinding the duplex
• Stabilizing the single strand
• Relieving the torque generated by unwinding
• Building a primer
• Assembling complementary strands
• Removing the primer
• Joining the Okasaki fragments

23
The One-Gene/One-Polypeptide Hypothesis

• The discovery that certain types of inherited
  diseases were prevalent in particular families
  led to the conclusion that
• These diseases were Mendelian traits which had
  resulted from changes in the hereditary
  information in an ancestor
• An example is alkaptonuria in which patients
  produce urine that contained homogentisic acid
  (alkapton)
• Such patients lacked the enzyme necessary to
  catalyze the breakdown of alkaptonuria
• Invariably it was concluded that genes specify
  particular enzymes
• This knowledge was clearly elucidated by Beadle
  and Tatum in their experiments involving the
  bread mold

24
The One-Gene/One-Polypeptide Hypothesis

• Beadle and Tatum were able to isolate mutant
  strains with defective form of that enzyme
• The mutations were always located at specific
  chromosmal sites and each enzyme had a different
  site
• Each mutant had a defect in a single enzyme
  caused by a mutation at a single site on the
  chromosome
• They concluded that genes produce their effects
  by specifying the structure of enzymes and each
  gene encodes the structure of one enzyme
• This relationship was termed by them the
  one-gene/one-enzyme hypothesis or
  one-gene/one-polypeptide